1. Field of the Invention
The present invention relates generally to recombinant strains of avian paramyxoviruses (APMV) and their use as oncolytic agents. In particular, the present invention is directed towards recombinant Newcastle disease virus (NDV) that exhibits enhanced oncolytic efficacy, and particularly to recombinant NDV that incorporates one or more additional therapeutic transgenes. The present invention also relates to methods of treating cancer by administering recombinantly-produced NDV to a patient. The present invention further relates to methods of providing targeted delivery of a recombinant NDV to specific sites in a patient, as well as methods of identifying recombinant NDV useful as oncolytic agents.
2. Description of Related Art
Naturally occurring or engineered oncolytic viruses (OVs) are emerging as novel tools for selective growth in and killing of a variety of tumor cells. OVs are multimodal therapeutics that can be engineered to have the tumor specificity of a small molecule, the potent cell killing ability of a chemotherapeutic agent, the ability to arouse the host immune system against tumor antigens, and an innate capacity to stimulate the production of host cytokines that have potential anticancer activity (Bell et al., “Oncolytic viruses: programmable tumour hunters,” Curr Gene Ther 2:243-54 (2002); Kruyt et al., “Toward a new generation of conditionally replicating adenoviruses: pairing tumor selectivity with maximal oncolysis,” Hum Gene Ther 13:485-95 (2002)). Among the OVs, the avian paramyxovirus Newcastle disease virus (NDV) is considered to be a very promising oncolytic agent (Reichard et al., “Newcastle disease virus selectively kills human tumor cells,” 7 Surg Res 52:448-53 (1992); Lorence, et al., “Complete regression of human fibrosarcoma xenografts after local Newcastle disease virus therapy,” Cancer Res 54:6017-21 (1994)).
The mechanism of viral oncolytic activity involves the induction of multiple caspase-dependent apoptotic pathways, and occurs despite normal IFN responses. Several viruses, including NDV, have been found to induce apoptosis in infected cells (Lam et al., “Apoptosis as a cause of death in chicken embryos inoculated with Newcastle disease virus,” Microb Pathog 19:169-74 (1995)). It has been shown that the tumoricidal activity of NDV on human monocytes is mediated by TRAIL, and TRAIL expression is independent of virus replication (Washburn et al., “TNF-related apoptosis inducing ligand mediates tumoricidal activity of human monocytes stimulated by Newcastle disease virus,” J Immunol 170:1814-21 (2003)). However, the exact cellular pathways involved in virus-induced apoptosis and the mechanistic basis of oncolysis are still incompletely understood.
Apoptosis is a multi-step, multi-pathway cell-death program that is inherent in every cell of the body. The apoptotic pathways leading to cell death can generally be divided into two nonexclusive signaling cascades (Igney et al., “Death and anti-death: tumour resistance to apoptosis,” Nat Rev Cancer 2:277-88 (2002)). In both pathways, cysteine aspartyl-specific proteases (caspases) that cleave cellular substrates are activated, and this leads to the biochemical and morphological changes characteristic of apoptosis. Both the intrinsic and the extrinsic pathways converge on downstream “executioner” caspases, mainly caspase-3, and caspase-6, and -7, which are responsible for the cleavage of structural cytoplasmic and nuclear proteins, with consequent cell collapse and death (Rathmell et al., “The central effectors of cell death in the immune system,” Annu Rev Immunol 17:781-828 (1999)). Activation of the death receptor and mitochondrion-associated death pathways are not mutually exclusive and these pathways may interact (cross-talk) at many levels.
The mitochondrion apoptotic pathway (intrinsic pathway) initiates with signaling from pro-apoptotic proteins from the BcI-2 family such as Bax, which trigger the release of cytochrome c in the induction phase. This in turn triggers the release of a second mitochondrion-derived activator of caspase (Smac/DIABLO), as well as apoptosis-inducing factor (AIF), and endonuclease G in the cytosol. (Du et al., “Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition,” Cell 102:33-42 (2000); Verhagen et al., “Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins,” Cell 102:43-53 (2000)). Cytosolic cytochrome c triggers the formation of a multimeric Apaf-1/cyt c/dATP/procaspase-9 protein complex termed the apoptosome, and the apoptosome then activates caspase-3, which in turns activates the caspase cascade and the degradation phase of apoptosis (Igney et al., supra). Caspase activation and the activity of already active caspases can be inhibited by the inhibitor of apoptosis proteins' (IAPs). Cytochrome c becomes a key regulator in the effector phase because once it is released from the mitochondria the cell is irreversibly committed to death.
The death receptor apoptotic pathway (extrinsic pathway) is initiated by binding of death activators (i.e., FasL, TNF) to their respective transmembrane death receptors (i.e., the tumor necrosis factor receptor (TNF-R) superfamily, which includes CD95 (Fas/APO-1), TNF-RI, DR3, DR4 (TRAIL-R1) and DRS (TRAIL-R2) receptors) (Ichikawa et al., “Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity,” Nat Med 7:954-60 (2001); Krammer, “CD95(APO-1/Fas)-mediated apoptosis: live and let die,” Adv Immunol 71:163-210 (1999); Nagata, “Apoptosis by death factor,” Cell 88:355-65 (1997)). Apoptosis initiated via death receptors involves the adaptor molecule FADD and subsequent proximity induced activation of caspase-8, an initiator caspase (Ha et al., “A novel family of viral death effector domain-containing molecules that inhibit both CD-95- and tumor necrosis factor receptor-1-induced apoptosis,” J Biol Chem 272:9621-4 (1997)). The activation of caspase-8, which is similar to caspase-9 in the intrinsic pathway, leads to activation of effector caspases and the degradation phase of apoptosis.
In addition, there is a third pathway that does not use caspases called the Apoptosis-Inducing Factor (AIF) pathway that occurs in neurons. Under an inducing signal, AIF located in the intermembrane space of the mitochondria is released and migrates into the nucleus. Once inside the nucleus it binds to DNA and triggers the destruction of DNA and the degradation phase of apoptosis.
Viruses that induce death receptor-dependent apoptosis include HIV (Miura et al., “Critical contribution of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to apoptosis of human CD4+T cells in HIV-1-infected hu-PBL-NOD-SCID mice,” J Exp Med 193:651-60 (2001)), measles virus (Vidalain et al., “Measles virus induces functional TRAIL production by human dendritic cells,” J Virol 74:556-9 (2000)), influenza A virus (Nichols et al., “Human lymphocyte apoptosis after exposure to influenza A virus,” J Virol 75:5921-9 (2001)), reovirus (Clarke et al., “Reovirus-induced apoptosis is mediated by TRAIL,” J Virol 74:8135-9 (2000)), and lyssa virus (Kassis et al., “Lyssavirus matrix protein induces apoptosis by a TRAIL-dependent mechanism involving caspase-8 activation,” J Virol 78:6543-55 (2004)). A number of viruses have been found to cause relocalization of proapoptotic mitochondrial proteins into the cytosol. Among these are HIV (Fern et al., “Mitochondrial control of cell death induced by HIV-1-encoded proteins,” Ann N Y Acad Sci 926:149-64 (2000)), influenza A virus (Chen et al., “A novel influenza A virus mitochondrial protein that induces cell death,” Nat Mod 7:130612 (2001)), HSV-I (Zhou et al., “Wild-type herpes simplex virus 1 blocks programmed cell death and release of cytochrome c but not the translocation of mitochondrial apoptosis-inducing factor to the nuclei of human embryonic lung fibroblasts,” J Virol 74:9048-53 (2000)), hepatitis B virus (Terradillos et al., “The hepatitis B: virus X protein abrogates Bcl-2-mediated protection against Fas apoptosis in the liver,” Oncogare 21:377-86 (2002)), reovirus (Kominsky et al., “Reovirus-induced apoptosis requires both death receptor- and mitochondrial-mediated caspase-dependent pathways of cell death,” Cell Death Differ 9:926-33 (2002)), and West Nile virus (Parquet et al., “West Nile virus-induced bax-dependent apoptosis.,” FEBS Lett 500:17-24 (2001)).
Apoptin (viral protein 3—VP3) is a gene product derived from the Chicken Anemia Virus (CAV), which appears to have innate-specific p53-independent, Bcl-2-enhanced pro-apoptotic activity. (Danen-van Oorschot et al., “Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells,” Proc. Natl. Acad. Sci. USA 94:5843-5847 (1997); Danen-van Oorschot et al., “Importance of nuclear localization of apoptin for tumor-specific induction of apoptosis,” J. Biol. Chem. 278:27729-27736 (2003); Oro et al., “The tumor specific pro-apoptotic factor apoptin (VP3) from chicken anemia virus,” Curr. Drug Targets 5:179-190 (2003); Huang et al., “Apoptin, a protein derived from chicken anemia virus, induces p53-independent apoptosis in human osteosarcoma cells,” Cancer Res. 55:486-489 (1995); Zhuang et al., “Differential sensitivity to Ad5 B1 B-21 kD and Bcl-2 proteins of apoptin-induced versus p53-induced apoptosis,” Carcinogenesis 16:2939-2944 (1995)). Recent studies with apoptin have shown that it induces G2/M arrest by targeting and inhibiting the anaphase-promoting complex/cyclosome (APC/C). Delivery systems for apoptin include mammalian expression plasmids, HIV-TAT protein transduction domain fusion, autonomous parvovirus and adenoviral vectors.
Newcastle disease virus (NDV), an avian paramyxovirus, is replication-competent in human tumor cells, intrinsically oncolytic, and is currently being tested for use as an oncolytic agent. NDV has been used in the clinic as an experimental oncolytic agent for more than 30 years (Csatary, “Viruses in the treatment of cancer,” Lancet 2:825 (1971); Lorence et al., “Newcastle disease virus as an antineoplastic agent: induction of tumor necrosis factor-alpha and augmentation of its cytotoxicity,” J Natl Cancer Inst 80:1305-12 (1988)). NDV is a member of the family Paramyxoviridae and has been assigned to the genus Avulavirus in the subfamily Paramyxovirinae (Mayo, “A summary of taxonomic changes recently approved by ICTV,” Arch Virol 147.1655-6 (2002)). It carries a serious respiratory and neurological disease in all species of birds, but infections in humans are rare, and any such infections typically result in no more than mild conjunctivitis.
NDV contains a single-stranded, negative-sense, nonsegmented RNA genome. The genomic RNA is 15,186 nucleotides in length (Krishnamurthy, et al., “Nucleotide sequences of the trailer, nucleocapsid protein gene and intergenic regions of Newcastle disease virus strain Beaudette C and completion of the entire genome sequence,” J Gen Virol 79(10):2419-2424 (1998)). The genomic RNA contains six genes that encode at least seven proteins (Steward et al., “RNA editing in Newcastle disease virus,” J Gen Virol 74 (Pt 12):2539-47 (1993)). The envelope of NDV contains two glycoproteins, the hemagglutinin-neuraminidase (HN) and fusion (F) proteins. The F glycoprotein mediates fusion of the viral envelope with cellular membranes (Choppin et al., “The role of viral glycoproteins in adsorption, penetration, and pathogenicity of viruses,” Rev Infect Dis 2:40-61 (1980)). In common with other paramyxoviruses, NDV also produces two additional proteins, V and W, from the P gene by alternative mRNAs that are generated by RNA editing (Griffin, “Neuronal cell death in alphavirus encephalomyclitis,” Curr Top Microbiol Immunol 289:57-77 (2005); Ichikawa et al., “Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity,” Nat Med 7:954-60 (2001); Lana et al., “Characterization of a battery of monoclonal antibodies for differentiation of Newcastle disease virus and pigeon paramyxovirus-1 strains.,” Avian Dis 32:273-81(1988); Rathmell et al., “The central effectors of cell death in the immune system,” Ann Rev Immunol 17:781-828 (1999); Sangfelt et al., “Induction of apoptosis and inhibition of cell growth are independent responses to interferon-alpha in hematopoietic cell lines,” Cell Growth Differ 8:343-52 (1997)).
NDV has been shown to be tumor-selective in replication, and cytolytic in infected tumor cells, while normal human cells are unaffected (Reichard et al., “Newcastle disease virus selectively kills human tumor cells,” 7 Surg Res 52:448-53 (1992)). One genetic defect that is common among tumor cells is diminished IFN “responsiveness” (Stojdl et al., “Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus,” Nat Mod 6:821-5 (2003)). NDV is a potent IFN inducer, and the tumor selectivity is therefore considered to be due to defective interferon response in tumor cells (Reichard et al., supra). Also, activating mutations in ras genes have been found in >30% of cancers, and constitutive ras pathway signaling brought about by oncogenic changes in upstream and downstream elements arises in an even greater proportion of human tumors. It has been demonstrated that some naturally occurring strains of NDV replicate well in tumors with ras gene activation, and are oncolytic in those tumors (Reichard et al., supra).
NDV strains are known to evoke cellular apoptosis (Lam et al., “Apoptosis as a cause of death in chicken embryos inoculated with Newcastle disease virus,” Microb Pathog 19:169-74 (1995); Washburn et al., “TNF-related apoptosis-inducing ligand mediates tumoricidal activity of human monocytes stimulated by Newcastle disease virus,” J Immunol 170:1814-21 (2003)). It has been suggested that TNF-α might be involved in the tumoricidal activity of NDV-activated murine macrophages and human peripheral blood mononuclear cells (Lorence et al., “Newcastle disease virus as an antineoplastic agent: induction of tumor necrosis factor-alpha and augmentation of its cytotoxicity,” J Natl Cancer Inst 80:1305-12 (1998); Washburn et al., supra). It is also claimed that the cell-to-cell contact killing of tumor cells by NDV-stimulated macrophages is mediated by TRAIL (Washburn et al., supra). Most of these studies tested the apoptotic response of NDV-activated human cells on human tumor cells. On the other hand, direct infection studies with NDV strain MTH/68 in PC12 rat phaeochromocytoma cells indicated that major mitogen-activated protein kinase pathways (including the stress inducible c-Jun N-terminal kinase pathway and p38 pathway) or mechanisms regulated by reactive oxygen species have no role in virus-induced apoptosic cell death (Fabian et al., “Induction of apoptosis by a Newcastle disease virus vaccine (MTH-68/H) in PC12 rat phaeochromocytoma cells,” Anticancer Res 21:125-35 (2001); Stojdl et al., “Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus,” Nat Mod 6:821-5 (2000)).
U.S. Pat. No. 6,896,881 discloses compositions and methods for treating a patient having a tumor, in order to reduce tumor size, by administering replication-competent Paramyxoviridae virus comprising two or more of a) a nucleic acid sequence encoding a heterologous polypeptide that is detectable in a biological fluid of the patient, where detection of the heterologous polypeptide is indicative of Paramyxoviridae virus growth in the patient and reduction in tumor size; b) a recombinant F protein, H protein, or M protein of Paramyxoviridae virus that increases fusogenicity of virus with cells; c) a nucleic acid sequence encoding a cytokine; and d) a Paramyxoviridae virus that is specific for cells of the tumor. The patent provides examples based only on the use of a recombinant measles virus.
U.S. Pat. No. 6,428,968 discloses compositions and methods for killing tumor cells in a patient, including administering both a chemotherapeutic agent and an oncolytic virus (other than an adenovirus) to a patient who has tumor cells. The agent and virus exhibit oncolytic activities that are at least additive, and that may be synergetic. The oncolytic virus may be a herpes simplex virus (type 1 or 2), a vaccinia virus, a vesicular stomatitis virus, or a Newcastle disease virus. The compositions and kits comprise a chemotherapeutic agent and an oncolytic virus (other than an adenovirus), either in admixture or separately.
U.S. Published Application No. 2004/0131595 discloses use of a negative-stranded RNA virus to treat a mammalian subject having a carcinoid tumor. The virus may be a Paramyxovirus, and may be a Newcastle disease virus.
U.S. Published Application No. 2003/0165465 discloses viruses that are able to replicate and kill neoplastic cells that have a deficiency in the IFN-mediated antiviral response. Such viruses may be used in treating neoplastic diseases, including cancer and large tumors. RNA and DNA viruses, including Paramyxoviruses, such as Newcastle disease virus, are stated to be useful in this regard.
U.S. Published Application No. 2003/0040498 discloses oncolytic activity of RNA-based vectors derived from poliovirus, termed replicons, which are genetically incapable of producing infectious virus. The replicons cytopathic in vitro for human tumor cells originating from brain, breast, lung, ovaries and skin (melanoma). Injection of replicons into established xenograft flank tumors in scid mice resulted in oncolytic activity and extended survival. Inoculation of replicons into established intracranial xenografts tumors in scid mice resulted in tumor infection and extended survival. Histological analysis was conducted in order to demonstrate that replicons infected tumor cells at the site of inoculation, and then diffused to infect tumor cells which had metastasized from the initial site of implantation.
With the availability of a reverse genetics system for NDV, it is now possible to manipulate the genome of NDV, engineer additional genes, and retarget the virus to specific receptors (Krishnamurthy et al., “Recovery of a virulent strain of Newcastle disease virus from cloned cDNA: expression of a foreign gene results in growth retardation and attenuation,” Virology 278:168-82 (2000); Huang et al., “High-level expression of a foreign gene from the most 3′-proximal locus of a recombinant Newcastle disease virus,” J Gen Virol 82:1729-36 (2001); Bian et al., “Tumor-targeted gene transfer in vivo via recombinant Newcastle disease virus modified by a bispecific fusion protein,” Int J Oncol 27:377-84 (2005)). However, there is still a need in the art for compositions comprising oncolytic viruses, such as NDV, and methods of using them to treat cancer in patient suffering therefrom. There is particularly a need in the art for genetically-engineered oncolytic viruses, such as NDV, which incorporate additional therapeutic transgenes into their genomes. Such genetically-engineered oncolytic viruses may be used in accordance with methods for providing targeted delivery of the oncolytic viruses to specific sites and/or specific tumors within the body of a patient.